Int. Res J Pharm. App Sci.

, 2014; 4(1):24-28 ISSN: 2277-4149

International Research Journal of Pharmaceutical and Applied

Sciences (IRJPAS)
Available online at www.irjpas.com
Int. Res J Pharm. App Sci., 2014; 4(1):24-28

Research Article

CHEMICAL SCREENING AND ANAESTHETIC ACTIVITY OF PHALLUSIA ARABICA

SAVIGNY, 1816
Kohila Subathra Christy H1, Jothibai Margret R2* and Meenakshi VK3*
1
Department of Chemistry, A.P.C. Mahalaxmi College for Women, Tuthukoodi, Tamil Nadu, India.
2
Department of Chemistry, Popes College, Sawyerpuram, Tuthukoodi, Tamil Nadu, India.
3
Department of Zoology, A.P.C. Mahalaxmi College for Women, Tuthukoodi, Tamil Nadu, India.

*Corresponding Author: Kohila Subathra Christy H ; Email: apcm.research@gmail.com

Abstract: Phallusia arabica is a simple ascidian belonging to the family Ascidiidae. It was extracted successively using different
solvents such as petroleum ether, benzene, methylene chloride, chloroform, ethanol, and water. Screening indicates the presence
of alkaloids, terpenoids, steroids, coumarins, tannins, saponins, flavonoids, quinones, anthroquinone, phenols, aromatic acids,
catechins, proteins, carbohydrates and lipids. Ethanolic extract of Phallusia arabica was subjected to anaesthetic activity by
intracutaneous wheal method. The extract, when administered at a dose of 20 and 40% caused highly significant anaesthetic
activity when compared to the standard drug xylocaine (0.5 & 1%). The mean sleeping time and percentage relaxation of muscle
was also highly significant in the extract treated groups compared to the standard drug, Aminobarbitone.
Key words: Phallusia arabica, Chemical screening, Anaesthetic, Xylocaine, Aminobarbitone

INTRODUCTION: Collection of animal material:

Pain is a physiologic response that has been defined in animals
as an aversive sensory experience caused by actual or potential
injury that elicits progressive motor and vegetative reactions,
resulting in learned avoidance, modifying species specific
behaviour1. Marine organisms are a rich source of structurally
novel and biologically active metabolites. Ascidians which are
marine organisms rank second with promising source of
drugs2. Most of the ascidians are utilized as food in various
countries and they are known to produce secondary
metabolites which prevent bio-fouling and this can be
considered as a kind of autogenic protection 3. The number of
natural products isolated from marine biota increase rapidly
and now exceeds with hundreds of new compounds being
discovered every year 4,5. A large proportion of these natural
compounds have been extracted from marine resources,
especially sponges, ascidians, bryozoans and molluscs and
some of them are currently in clinical trials6. This search for
new metabolites has resulted in the isolation of more or less
10,000 metabolites7.
Chemical screening strategy can benefit from the use of
standards or target molecules which can be rapidly
characterized by diagnostic parameters8. Qualitative and
Quantitative estimation of the chemical constituents will be
very much useful in the standardization of drugs. In India only
very few works has been carried out in ascidians. A review of
literature reveals that mainly taxonomical, biofouling,
antibacterial, antimitotic, antimicrobial studies, infrared and
GCMS studies of a few species of ascidians are available9-14.
Hence an attempt has been made to perform chemical
screening and anaesthetic study of Phallusia arabica Savigny
in this present work.
EXPERIMENTAL SECTION:
Kohila Subathra CH et al., 2014
Collection of Phallusia arabica was carried out from Tuticorin
coast by SCUBA diving. It was identified, authenticated and a
voucher specimen AS 2276 has been deposited in the National
Collections of Ascidians in the Museum of the Department of
Zoology, A.P.C. Mahalaxmi College for Women, Tuticorin-
628 002 (Figure 1).
Figure 1: Phallusia arabica Savigny, 1816
Preparation of extract:
The whole animal was dried in shade and homogenized to get
a coarse powder which was stored in an airtight container and
used for further investigations. 100g of powdered animal
material was extracted with petroleum ether, benzene,
methylene chloride, chloroform, ethanol and water using
soxhlet apparatus.
Experimental animal:
Mature adult male wistar albino rats weighing about 120-180 g
were selected for the study. They were maintained in a well
ventilated animal house with constant 12 hours of darkness
and 12 hours of light schedule. Clean water and standard pellet
diet (Hindustan Lever Ltd., India) were given ad libitum.
24
Int. Res J Pharm. App Sci., 2014; 4(1):24-28 ISSN: 2277-4149

Chemical screening: injection was noted. The normal responses of the animals were
The petroleum ether, benzene, methylene chloride, observed first by applying pin pricks in the midline. Ten pin
chloroform, ethanol, and water extracts of Phallusia arabica pricks were then given uniformly every five minutes at an
were screened for various chemical constituents such as interval of four seconds on the wheal areas. The responses
alkaloids, terpenoids, steroids, coumarins, tannins, saponins, were recorded up to 30 min. A localized skin twitch, usually
flavonoids, quinones, anthroquinone, phenols, aromatic acids, accompanied by squeak, was considered as the normal
catechins, proteins, carbohydrates and lipids using standard response to pin prick. When the animal failed to respond either
procedures15,16. by twitching of the muscle or squeaking following a pin prick,
a negative response was recorded19,20.
Acute toxicity studies:
Acute oral toxicity studies were performed to determine General anaesthetic activity:
minimum sub lethal doses of the animal extract17. During the Aminobarbitone induced sleeping time and muscle
24 hour observation period no adverse effect or mortality was relaxation:
observed up to 500 mg/kg body weight of ethanolic extract. The animals were divided into four groups of six each. Group
Hence 100, 200 and 400 mg/kg bw of the ethanolic extract I was given 10 mg/kg of standard drug, Aminobarbitone and
was selected for the study of anaesthetic activity. II, III and IV received 100, 200 and 400 mg/kg bw of the
extract. The mean sleeping time and muscle relaxation (% of
Local anaesthetic activity: rats unable to grasp the board with fore paws) were noted 21.
Intracutaneous wheal method:
The local anaesthetic activity of the ethanolic extract of RESULTS AND DISCUSSION:
Phallusia arabica was studied by intracutaneous wheal Alkaloids, terpenoids, steroids, tannins, flavonoids, quinones,
method in albino rats18. One day prior to the study, four cm proteins, lipids and carbohydrates have been observed in all
area of hair on four different areas on the back of albino rats the extracts whereas coumarins was not found in any of the
near the midline were clipped and removed. The animals were extracts Table 1. Saponins were present in petroleum ether,
divided into five groups of six each. Group I, II were treated benzene, methylene chloride and chloroform extracts. Except
with 0.5 and 1.0 % standard drug, xylocaine and III, IV, V petroleum ether extract all other extracts contain
received 10, 20, 40% of the ethanolic extract. The drugs were anthroquinone. Phenols and aromatic acids were observed in
injected intracutaneously in equal volumes of 0.2 ml into the methylene chloride, ethanol and water. Catechins were
shaved areas, wheals were marked with ink and the time of detected in methylene chloride and ethanol extracts.
Table 1: Chemical screening of Phallusia arabica
(40 - 60 0C)

Chloroform
Petroleum

Methylene
Chloride
Benzene

Ethanol

Water
Ether

S. No. Chemical Constituents

1. Alkaloids + + + + + +
2. Terpenoids + + + + + +
3. Steroids + + + + + +
4. Coumarins - - - - - -
5. Tannins + + + + + +
6. Saponins + + + + - -
7. Flavonoids + + + + + +
8. Quinones + + + + + +
9. Anthraquinone - + + + + +
10. Phenols - - + - + +
11. Catechins - - + - + -
12. Aromatic acids - - + - + +
13. Proteins + + + + + +
14. Lipids + + + + + +
15. Carbohydrates + + + + + +
Key: + Present; - Absent.

The results of the anaesthetic study shows that the extract
produced significant anaesthesia in 20% (81.66%) and 40%
(90%) dose when compared to standard drug Xylocaine (0.5%
(43.33%) and 1% (53.33%) Table 2 and Figure 2. The
negative responses of the extract treated groups showed a
highly significant increase when compared to that of the
standard. An increased concentration of the test drug produced
an elevated local anaesthetic activity. Dose dependent
proportionate increase in sleeping time and muscle relaxation
were noted. Maximum sleeping time of 198 5.84 min and
214 7.35 min were observed in group III and group IV
respectively compared to that of standard aminobarbitone 179
4.80. Muscle relaxation was also found to be high in group
III (75%) and group IV (85%) Table 3 and Figure 3. Some

Kohila Subathra CH et al., 2014 25

Int. Res J Pharm. App Sci., 2014; 4(1):24-28 ISSN: 2277-4149

workers have reported that the local anaesthetic property could
be due to the presence of alkylamides22. Ascidians are
renowned for their overwhelming bias towards the production
of nitrogenous secondary metabolites23. Rita and coworkers
reported that alkaloids and saponins may be responsible for
anaesthetic activity24. The preliminary chemical screening of
the ethanolic extract of Phallusia arabica revealed the
presence of alkaloids, flavonoids, terpenoids, anthraquinone
and one or more of the chemical components may be
responsible for the activity. Similar results have been reported
using the methanolic extract of Phallusia nigra25.

Table 2: Local anesthetic effect of Phallusia arabica

Known Number of negative responses over time (min) Total
drug/Extract/gr Dose out of Anaesthesia
oup (%) 0 5 10 15 20 25 30 60 (%)

Xylocaine - I 0.5 0 8 4 7 4 2 1 26 43.33

Xylocaine - II 1.0 0 7 8 6 3 4 2 32 53.33
Extract - III 10 0 7 6 7 6 5 7 38 63.33*
Extract - IV 20 0 9 8 7 8 6 9 49 81.66**aa
Extract - V 40 0 10 9 9 6 7 10 54 90.00**aa
Data represented as mean of 10 observations, (n6). Significance between control low dose and extract treated groups * p <0.05;
** p <0.01. Significance between control high dose and extract treated groups a p <0.05; aa p <0.01.

100
Anaesthesia %
80
Anaesthesia (%)

60
40
20
0
I II III IV V
Groups Treatment

Figure 2: Local anaesthetic effect of Phallusia arabica

Table 3: The general anaesthetic effect of Phallusia arabica

Muscle Relaxation (% of rats
SleepingTime
unable to grasp board with fore
Groups Drug/Dose (mg/Kg bw) (MinSD)
paws)
Mean Time
I Aminobarbitone 10 1794.80 60%
II 100 1824.55ns 40%
III 200 1985.84** 75%
IV 400 2147.35*** 85%
Data represented as mean S.E.M, (n6). Significance between control and extract treated groups. * p <0.05; ** p <0.01; *** p
<0.001.

Kohila Subathra CH et al., 2014 26

Int. Res J Pharm. App Sci., 2014; 4(1):24-28
100
% of Muscle Relaxation
80
60
40
20
0
Group I
Figure 3: The general anaesthetic effect of Phallusia arabica
CONCLUSION
It can be concluded, that the ethanolic extract of Phallusia
arabica has significant anaesthetic properties. However further
detailed study is needed in order to understand the active
principle and the mode of action responsible for this activity.
Acknowledgement:
The authors thank the University Grants Commission,
Hyderabad for financial assistance No: F. MRP-4218/12
MRP/UGC-SERO and to Dr. S. Sampath Raj, Samsun Clinical
Research laboratory, Thirupur for providing assistance to carry
out the pharmacological studies.
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